Staphylococcus aureus (S. aureus) is one of the most frequent causes of bacterial infections in the U.S. and is responsible for diverse types of infections ranging in severity from mild to fatal in both hospital and community settings. In the community, S. aureus typically causes skin-and-soft-tissue infections with an estimated 12 ? 13 million outpatient visits per-year (in the U.S.) but can also cause severe manifestations including necrotizing fasciitis and sepsis in individuals with no underlying risk. One of the most clinically significant aspects of S. aureus infections is the high prevalence of drug resistance and the innate ability for S. aureus to develop resistance to antibiotics making it very difficult to develop therapeutics that will have potential for long-term efficacy on S. aureus. The research proposed in this application will broaden our understanding of host- pathogen interactions, providing the foundation for future studies aimed at the intelligent design of novel vaccines and therapies to treat bacterial infection. To that end, this project will study how S. aureus uses the SaeR/S two-component gene regulatory system to evade innate immunity. The SaeR/S system is essential for evasion of neutrophil killing; however, exact mechanisms dependent on SaeR/S resulting in neutrophil dysfunction are not defined. Three specific Aims will test the hypothesis that the SaeR/S-mediated up- regulation of S. aureus virulence genes undermines an effective neutrophil response that would normally clear infection. In Aim One, host pathogen RNA expression via RNA-seq will be used to determine the direct influence of SaeR/S-regulated bacterial genes on human neutrophil gene expression as well as resolve the individual contribution of SaeR/S components on neutrophil and pathogen gene expression simultaneously. In Aim Two, we will determine how SaeR/S-dependent changes in neutrophil human myeloperoxidase (MPO) activity and the production reactive oxygen species (ROS) alters neutrophil apoptosis and neutrophil extracellular trap (NET) formation. In Aim Three, we will utilize a novel ex vivo model of neutrophil priming by stimulated PBMCs to determine the role of initial SaeR/S-mediated immune cell interactions on subsequent neutrophil function. These ex vivo primary human cell studies will be complemented with murine models of infection to thoroughly evaluate these immune responses. Findings from experiments outlined in this proposal will improve our overall understanding of host-pathogen interactions by specifically characterizing novel SaeR/S-mediated mechanisms used by S. aureus to overcome an effective neutrophil response. Collectively this research will address our long-term objective to characterize reciprocal response pathways between host and pathogen that will highlight key factors needed for both the pathogen to cause infection and for the host to resolve disease. Results will provide valuable information for vaccine and immunotherapeutic development.